Assay validation of phosphorylated S6 ribosomal protein for a pharmacodynamic monitoring of mTOR-inhibitors in peripheral human blood.
Cytometry B Clin Cytom
; 82(3): 151-7, 2012 May.
Article
em En
| MEDLINE
| ID: mdl-22213594
ABSTRACT
BACKGROUND:
Therapeutic drug monitoring (TDM) of immunosuppressive drugs after organ transplantation is based on measuring blood levels alone, which often results in under- or over-immunosuppression. Previous studies have shown the potential of measuring pharmacodynamic drug effects for TDM, but assessment of biomarkers for individual drugs is still not clinical routine. Therefore, we validated a specific assay to measure the pharmacodynamic effects of mammalian target of rapamycin (mTOR)-inhibitors on phosphorylated S6 ribosomal protein (p-S6RP), a downstream target of mTOR.METHODS:
Clinical relevant concentrations of sirolimus (SRL, 0.9-91.4 µg/L), cyclosporine A (CsA, 75.1-1202 µg/L), mycophenolate acid (MPA, 0.08-3.2 mg/L), or dexamethasone (DEX, 0.5-200 ng/mL) were added to whole-blood from healthy volunteers. Activated whole-blood was analyzed by phospho-flow cytometry to measure p-S6RP in T cells.RESULTS:
Phospho-flow analysis revealed that SRL suppressed p-S6RP in human T cells in a dose-dependent manner with a half-maximal inhibitory concentration (IC(50)) at 19.8 nM and a maximal inhibitory effect (I(max) %) at 91.9%. Neither CsA, MPA, nor DEX inhibited mTOR-related S6RP-phosphorylation. Coefficient of variations from 0.03 to 0.05, 0.12 to 0.25, and 0.14 to 0.38 for intra-, interassay, and interindividual variability respectively, showed robustness of our assay. Furthermore, samples can be stored at RT or 4°C up to 2 h after withdrawal.CONCLUSION:
We validated a robust whole-blood assay that allows the specific measurement of SRL- and everolimus-induced inhibition of T cells' function through detection of p-S6RP. Future studies in organ transplanted recipients will show if this assay has the potential to enhance a TDM for mTOR-inhibitor drugs in combination therapies.
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Base de dados:
MEDLINE
Assunto principal:
Linfócitos T
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Monitoramento de Medicamentos
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Proteína S6 Ribossômica
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Serina-Treonina Quinases TOR
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Imunossupressores
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article